A study was made to observe the association between the resistance to reinfection induced by irradiated metacercariae (MC) of Clonorchis sinensis and antigen specific Th1- and Th2-type cytokine productions in rats. Rats were infected with 20 MC of C. sinensis, previously exposed to a single dose of gamma irradiation, which varied from 0 to 100 Gy. All of them, single dose of 12 Gy showed higher IgG antibody titer with lowest worm recovery. Thus, 50 MC were used to challenge infection in rats previously infected with 20 MC irradiated at 12 Gy and the highest resistance to challenge infection was observed. The results of lymphocyte proliferation with specific antigen, ES Ag were shown no difference of proliferative responses as compared with primary and challenge infection at 12 Gy irradiation dose. In the case of cytokines production were observed that interferon (IFN-g) and interlukin (IL-2) were significantly enhanced, while IL-4 and IL-10 was almost unchanged to make comparison between primary and secondary infection at 12 Gy irradiation dose. In conclusion, the single dose of 12 Gy could be adopted for induction of the highest resistance to challenge infection. Up-regulation of Th1 type cytokines, IFN-g and IL-2 may be affected to develop vaccine by irradiated MC.

Liver fluke infection caused by Clonorchis sinensis is still an important public health problem in many countries of the West Pacific area. It is estimated that about 30 million people are infected annually with C. sinensis or Opisthorchis viverrini by eating uncooked fresh water fish in Southeastern Asia (Rim et al. 1981, Akai et al. 1994). Studies about effects of irradiation on infective stage of C. sinensis have been reported (Lee et al. 1989, Song et al. 1992, Duan et al. 1993), and reports show that suitable dose of irradiation on isolated metacercariae (MC) or un-isolated MC in fish can prevent infections. There are evidences from immunological studies that man and animals produce antibodies and cellular immunity after C. sinensis infections (Choi & Park 1987, Choi et al. 1990, Hagan et al. 1991, Akai et al. 1994, Quan et al. 2000, 2002). To the best of our knowledge no study was carried out on the association between the effects of gamma irradiation and immune responses in clonorchiasis. However, study on Schistosoma mansoni showed that high levels of protection have been achieved in mice against S. mansoni infection using gamma irradiated cercarial vaccines (Dean 1983, Coulson 1997). Also Th1- or Th2-type cytokines were reported to associate with protective immunity against various parasites (Stevenson & Tam 1993, Cetre et al. 1999, Koyama et al. 1999). In the present study, C. sinensis MC were first treated with different dose of gamma irradiation to select the suitable dose for the study of immune responses. Then resistance to reinfection induced by irradiated MC of C. sinensis was evaluatedand an association between resistance and Th1- and Th2-type cytokines were observed.

MATERIALS AND METHODS

Parasites and experimental animals - Sprague-Dawley (SD) rats (female, 8 wk old) were purchased (Samyook animal center, Osan-shi, Kyonggi-do, South Korea) and used for whole experimental process. Parasites C. sinensis MC collected from Pseudorasbova parva were used for infection of rats and rabbits. Male white rabbits (New Zealand white) about 1.5 to 2 kg were used to recover adult C. sinensis for collection of C. sinensis excretory-secretory antigen (ES Ag). All procedures involving animals and their care were in conformity with institutional guidelines that comply with national and international laws and policies.

Antigen preparation - C. sinensis MC were collected from P. parva and digested with artificial gastric juice, and orally administered to the experimental rabbits. Then adult worms were collected from rabbit liver after three month of infection. C. sinensis excretory-secretory antigen (ES Ag) was obtained by culturing living adult worm in RPMI 1640 medium supplemented with antibiotics at 37°C and 5% CO2. Culture medium was collected every two days and new medium was added to culturing living adult worm. Collected medium was centrifuged at 4°C, 10,000 rpm for 30 min, and the supernatant was used as ES Ag and stored at 70°C for use. Protein concentration of antigens was analyzed by the DC Protein Assay kit (Bio-Rad Lab, Hercules, CA, US) and stored at 70°C until used.

Study design - To determine the relationship between survival of worms and irradiation dose, rats were given irradiated MC of single dose that varied from 0 to 100 Gy. Then worm recovery and IgG antibody responses were measured. Thus, irradiation dose was selected with the highest titer of IgG and the lowest worm recovery. For the study of resistance to reinfection induced by selected irradiation dose and cytokine production, the experiment groups were designed as follows: uninfected control, rats infected with normal MC (non-irradiated MC), rats infected with irradiated MC, rats received challenge infection, previously infected with normal MC. Each group contained 10 rats.

Primary infection and challenge infection - In the experiments, rats were infected with 20 normal or 20irradiated MC in the primary infection. After one month, challenge infections were given with 50 normal MC via orogastric tube under light ether anesthesia (Quan et al. 2000, 2002).

Worm recovery and evaluation of protection - Worms were collected in the liver duct of rats in 4th week in the primary infection for the determination of irradiation dose. Worm recovery for evaluation of resistance was performed in 4th week after challenge infection. Protection was evaluated by comparing the adult worms recovered from experimental groups and control group.

Sera collection - Blood was collected from each rat in week 4 after primary infection during the selection of irradiation dose. Blood was allowed to clot at room temperature and stored at 4°C. After removal of the clot, the sera were centrifuged at 3000 rpm for 10 min and stored at 20°C until needed. Sera were used to measure IgG levels.

Enzyme-linked immunosorbent assay (ELISA) - The solid phase ELISA test was performed in a microtiter plate as described elsewhere (McLaren et al. 1978) with some modifications. Plates for IgG was coated with 5 µg/ml C. sinensis ES Ag. Serum samples were diluted 1:100. Peroxidase-conjugated goat affinity purified antibody rat IgG (Cappel, Durham, NC, US) in 1:1000 dilutions were used in duplicate wells. The reactions were stopped using 5 N H2SO4 and the OD at 490 nm was recorded using an ELISA reader (Molecular Devices,Menlo Park, CA, US).

Lymphocyte proliferation assay - For detection of lymphocyte proliferation and cytokine production, lymphocytes in mesenteric lymph nodes (MLN) were collected in the 4th week after primary or secondary infection. MLN were excised from rats and cell suspensions were aseptically prepared by squeezing the MLN between two sterile glass slides. Lymphocytes were pooled from 5 rats and cultured at a final concentration of 5 x 106 cells in 200 µl RPMI 1640 supplemented with 10% fetal bovine serum, 100 units of penicillin/streptomycin, 2 mM glutamine, 25 mM HEPES, 1% non-essential amino acid solution, 0.1% 5 x 10-2 M 2-mercaptoethanol and 1% sodium pyruvate (Invitrogen Co., Grand Island, NY, US). Cultures were performed in 96-well flat bottom culture plates for three days at 37°C in 93% air and 5% CO2. Cells were stimulated with ES Ag at 25 µg/ml, or phytohemaglutinin (PHA) at 10 µg/ml final concentration. Each test was performed three times. Cells were pulsed for the last 18 h with 0.5 µCi/well [3H]thymidine (Amersham Co., Arlington Heights, IL, US) and then harvested on glass fibre filters with a semi-automatic cell harvester (Skatron, Norway). Incorporated radioactivity was measured in a liquid scintillation counter (LKB 1214 Racbeta) and expressed as geometric means after subtracting the background counts (DCPM).

Cytokine analysis - For detection of cytokine production, triplicate cultures of 5 x 106 cells as described above were incubated with antigens in 96-well culture plates at 37°C for three days, in 5% CO2. The supernatants of three wells were pooled following centrifugation and stored at 70°C. The concentrations of interferon (IFN-g) and interleukins (IL-2, IL-4, IL-10) were determined in the culture supernatants by rat Cytoscreenä Immunoassay Kits (Biosource, California, US).

Statistical analysis - Statistical significant differences from all groups were carried out using ANOVA of SAS system. A value of P < 0.05 was considered significant. Cytokine productions were carried out using pooled cells from five rats and as such were not subject to statistical analysis.

RESULTS

Relationship between survival of worms and irradiation dose - Some different number of adult worms was recovered from rats infected with 20 irradiated or non-irradiated MC of C. sinensis after four weeks of infection. As shown in Fig. 1, worm burdens were reduced following increasing dose of radiation. Worms were recovered when MC were irradiated at 1, 3, 5, 7, 10, and 12 Gy irradiation dose, including non-irradiated control (0 Gy) (Fig. 1), whereas we could not observe survival worms at 15, 20, 40, 60, and 100 Gy irradiation dose.

Antibody responses in rats infected with irradiated MC - All rats (n = 9/each dose) had been infected with MC irradiated with a range of dose (1-20 Gy) showed significant increases in IgG antibody responses from 1 to 12 Gy range compared with the uninfected control (Fig. 2, P < 0.01). But, IgG antibody responses are low level in rats infected with 15 and 20 Gy irradiated MC. These data are consistent with the above that adult worms were recovered from the range of 1 to 12 Gy. The high antibody responses were detected in rats infected with 1 and 12 Gy irradiated MC, including non-irradiated (0 Gy) MC.

Resistance to reinfection - For detection of resistance to reinfection, rats were primarily infected with 20 irradiated MC ranging from 7 to 15 Gy. Then challenge infections were performed in rats using 50 non-irradiated MC after four weeks of primary infection. Fig. 3 showed that the number of C. sinensis adult worms recovered from rats was gradually decreased from range of 7 to 12 Gy, in which the highest resistance to reinfection was found in 12 Gy irradiation dose. Whereas high worm burdens were recovered in rats when challenge infection was carried out after primary infection with 15 Gy irradiated MC (Fig. 3). This result is the same as when rats only received challenge infection. And because these data are consistent with high IgG antibody responses in rats infected with 12 Gy irradiated MC, we assumed that MC died, and therefore lost ability to infect rats when MC were irradiated with dose over 15 Gy.

Thisstudy is about the resistance induced by irradiated MC of C. sinensis. Previous studies on parasite C. sinensis showed that 50 Gy irradiation doses on isolated MC or 150 Gy on fish is an effective control measure that can be used in preventing MC (Lee et al. 1989, Song et al. 1992). Present study used different doses that varied from 1 to 100 Gy to select optimal dose, in which worms remained the least in rats to reduce damage of liver tissue (Fig. 1). Thus, 12 Gy irradiated MC was selected in which rats showed high level of IgG antibody responses to the same level as those infected with non-irradiated MC (Fig. 2). Rats showed no antibody responses and no worms were recovered from 15 to 100 Gy irradiation dose compared to uninfected control and non-irradiated rat control. It may be that no worms survived on these irradiation doses. So an attempt to induce resistance was carried out on 12 Gy irradiation doses, in which worm burdens were significantly decreased after challenge infection (Fig. 3).

Little is known about the mechanisms by which protection against parasite infection is induced by irradiation-attenuated vaccines, but several studies have been performed including disruption of protein synthesis, changes in carbohydrate expression (Wales & Kusel 1992), alteration of parasite antigens, alteration of expression of cathepsin-B protease and WGA- and Con A-specific sugars which may be detrimental to parasite invasion and contribute to the protective immune responses generated in the host (Creaney et al. 1996). Maybe 12 Gy irradiation dose can induce maximum changes described above correlated with the capacity of the irradiated larvae (highest level of antibody responses) to protect rat against subsequent challenge with non-irradiated MC. Different levels of antibody responses from different doses need further study.

Our findings in this study indicated that ES Ag specific stimulated proliferative responses and cytokines productions were elicited. When the cytokines production compared to infection with 12 Gy irradiated MC and challenge infection with non-irradiated MC at ES Ag group, IFN-g and IL-2 were significantly enhanced. This result indicates that irradiated MC like attenuated vaccine is able to induce Th1-type cytokines (IFN-g and IL-2) and these cytokine effect to protection against C. sinenis infection. Resistance to different parasitic diseases has been associated with the both of Th1- or Th2-type immune responses. Different T-helper subsets appear when different parasite is presented in a host, or if the duration of infection is altered. S. mansoni infection can drive Th2 responses in rats or in mice and it favors protective immunity against reinfection (Cetre et al. 1999). Trichinella spiralis infection shows mixed Th1/Th2 response in rats (Stewart et al. 1999), Trichuris muris shows Th2-type response in mice involved in protective immunity (Koyama et al. 1999). Chronic infection with Taenia crassiceps is characterized by high levels of production of both Th1 and Th2 cytokines in mice (Spolski et al. 2000). Mice showed low parasite numbers when receiving recombinant cytokines IFN-g and IL-2, whereas significant increases in parasite loads were found when mice receiving IL-10 (Terrazas et al. 1999). It is well known that Th1-type response helps to eliminate intracellular microorganisms, whereas a Th2-type response specializes in the control of extra-cellular pathogens (Cox & Liew 1992, Stevenson & Tam 1993, Reiner & Locksley 1993). Development of an inappropriate immune response can be ineffective and even pathogenic to the host (Romagnani 1997). In Leishmania major infection, IL-10 administration leads to a higher parasite persistence in a mouse model and influenced the outcome of the disease by modifying the inflammation and local cell recruitment at the site of parasite penetration (Viana da Costa et al. 2002).

In conclusion, the single dose of 12 Gy could be adopted for induction of the highest resistance to challenge infection of C. sinensis. After primary infection with 12 Gy irradiated C. sinensis, reinfection with non-irradiated C. sinensis is induced high levels of production of IFN-g and IL-2. These Th1-cytokines were involved in protecting against C. sinenis reinfection.